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The isolation and properties of a flavoenzyme from rat liver is described. The enzyme, for which the name DT diaphorase is suggested, catalyzes the oxidation of reduced di- and triphosphopyridine nucleotide at equal rates. As electron acceptor, dyes such as 2 ,6-dichlorophenolindophenol, as well as various naptho- and benzoquinones without long carbon-chain substituents, are the most active. The reactivity of the enzyme with vitamin K1, vitamin K2, coenzyme Q10, cytochrome c and cytochrome b5 is negligible, and with ferricyanide and methylene blue relatively low. The enzyme is markedly activated by bovine serum albumin, polyvinylpyrrolidone and by certain non-ionic detergents, whereby both its maximal velocity and its substrate affinity are increased. Dicoumarol very strongly inhibits DT diaphorase, the Ki value being of the order of 10−8M. The inhibition is competitive with regard to the reduced pyridine nucleotides, but independent of the concentration of the electron acceptor. The enzyme also is inhibited by certain sulfhydryl reagents, thyrozine analogues, and flavin inhibitors. These properties of DT diaphorase are compared with those of other pyridine nucleotide oxidizing flavoenzymes. The conclusion is reached that DT diaphorase very probably is identical with the phylloquinone or vitamin K reductase described by Martius et al. The possible function of DT diaphorase is discussed.